Alcoholic liver disease (ALD) is one of the most common forms of chronic liver disease in the United States. Chronic ethanol consumption results in lipid accumulation in the liver and increased cell stress leading to inflammation and liver damage. Indeed, the crosstalk between hepatocytes and non-parenchymal cells - including Kupffer and hepatic stellate cells (HSCs) - is crucial to this process. However, the molecular mechanisms and signaling pathways involved in the crosstalk between lipid overloaded hepatocytes and non-parenchymal cells during ethanol- induced liver injury remain poorly understood. This issue is significantly important, as the identifying factor, or factors, that communicate stress signals from hepatocytes, and may initiate and perpetuate the inflammatory reaction responsible for liver damage and disease progression to steatohepatitis and cirrhosis may have a tremendous biomedical impact. We recently revealed that damaged hepatocytes release extracellular vesicles (EVs) and these EVs circulated in the blood in models of ALD and Nonalcoholic fatty liver disease (NAFLD). EVs have evolved as key cell-to-cell communicators and are comprised of non-coding RNAs such as microRNAs (miRNAs), messenger RNAs, proteins, and lipids, all of which are derived from parental cells. Data from our laboratory, as well as others, have demonstrated that EVs are efficiently internalized into target cells and transfer their miRNA cargo, which is a key mechanism by which EVs modulate protein translation in target cells. Thus demonstrating that encapsulated miRNAs in EVs (EV-miRNAs) are functional extracellular RNAs that mediate disease progression. Based on these preliminary data we propose the CENTRAL HYPOTHESIS that EV-miRNAs released from damaged hepatocytes will contribute ALD progression through macrophage and HSC activation. To investigate this hypothesis our application has following SPECIFIC AIMS. FIRST we will confirm and extend our preliminary results and establish the characteristics of EVs encapsulating miRNAs (EV-miRNAs) that are released from hepatocytes damaged from alcohol exposure. SECOND we will explore EV-miRNA function on macrophage and HSC biology using various in vitro assays and observe how the interplay relates to the progression of ALD in vivo. To address these central issues, we will get support from USC for intragastric feeding mild ASH or AH mice, from UCSD Institution for Genomic Medicine for microRNA sequencing, and from UCSD Prof. Dowdy for miRNA delivery. These exploratory studies will break new ground and may uncover key roles for extracellular miRNAs in mediating liver damage during ALD, as well as serve as potential novel biomarkers and therapeutic targets.